Antibiotic-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa are among the most common causes of life-threatening infections in the world. Unfortunately, these infections are increasingly difficult to treat due to the recent explosion in disease caused by methicillin-resistant S. aureus (MRSA) and multi-drug resistant P. aeruginosa (MDRPA). Given their high incidence of causing severe, drug-resistant infections, novel approaches to prevent or treat infections caused by MRSA and MDRPA would have enormous beneficial impact on U.S. and global health. We recently discovered that human platelet microbicidal proteins are microbicidal chemokines, called "kinocidins" to reflect their dual functions. Kinocidins share a consensus structural motif (the ?-core) with classical antimicrobial peptides, and exert rapid and potent microbicidal effects versus pathogens that access the bloodstream, including MRSA and MDRPA. However, kinocidins differ markedly in their overall structural configuration as compared to classical antimicrobial peptides (eg., defensins), the majority of which are cytotoxic or inactivated when released into the bloodstream. Kinocidins are much less cytotoxic to human vascular endothelial cells or erythrocytes in vitro as compared with defensins. In exploring the structural basis for their minimal host toxicity, we made the highly promising discovery that kinocidins disassemble in the context of active infection through cleavage by proteases generated by virulent pathogens, or that emanate from tissues infected by these organisms. Based on this structure-activity paradigm in human kinocidins, we engineered a novel class of polypeptides designed to achieve three critical functions: i) activate in response to signals emanating from virulent organisms or tissues infected thereby; ii) exert potent microbicidal efficacy in relevant contexts, including blood and blood matrices; and iii) have little or no concomitant host cell toxicity as compared with classical antimicrobial peptides. These resulting molecules are termed context-activated protides. We have demonstrated proof of principle by engineering, expressing, and documenting that prototype protides exert 50-fold greater efficacy against MRSA expressing V8 protease than a V8-deficient, avirulent counterpart. Moreover, kinocidin modules retain potent microbicidal activity versus MRSA and MDRPA in whole blood and plasma. Based on these exciting preliminary data, we will assess the feasibility of novel context-activated protides targeting severe MRSA and MDRPA infections. To overcome problems that have contributed to recent failures of antimicrobial peptide therapeutic strategies, and lay the foundations for advancement of context-activated protide technology, our goals for Phase I of the current STTR application are: 1) To generate a combinatorial library of novel context-activated protides using accelerated evolution; 2) To prioritize lead candidate protides for optimal therapeutic index in highly relevant biomatrix assays; and 3) To validate the efficacy of a lead candidate protide in established models of invasive infection. Context-activated protides exploit structural and mechanistic signatures of host defense peptides optimized over millions of years by Nature. This Phase I STTR project is a logical extension of these discoveries, and will validate the feasibility of context-activated protides that target MRSA and MDRPA infections. Outcomes will focus development of lead candidate protides in Phase II of the STTR, to define efficacy against diverse MRSA and MDRPA strains, establish GMP, complete pre-clinical toxicity studies, and submit an IND for phase I clinical trials. This platform technology may also enable otherwise toxic antimicrobial peptides, such as defensins, as therapeutic agents. These advances would represent major breakthroughs in the prevention and treatment of these common and increasingly difficult-to-treat infections. [unreadable] [unreadable] [unreadable]